Composition to enhance HDL cholesterol and to decrease intima-media thickening in animals and humans and a method for its preparation

ABSTRACT

A method of producing a product to correct hypercholesterolemia including pulping fruits of  Emblica officinalis  with demineralized water to create a slurry. The slurry is treated with pectinase. The pectinase-treated slurry is filtered to create a solution. The solution is concentrated to create a product. A product having an extract of  Emblica officinalis  for prophylactic and for therapeutic treatment of coronary diseases, atherosclerosis, hypothyroidism and hyperthyroidism.

This is a Divisional of U.S. application Ser. No. 13/374,931, filed Jan.24, 2012, which is a divisional of Ser. No. 12/805,191, filed Jul. 16,2010, which is a Divisional of U.S. application Ser. No. 11/643,788,filed Dec. 22, 2006, which is a continuation of U.S. application Ser.No. 11/111,798, filed Apr. 22, 2005, which is a continuation ofInternational Application PCT/IN2003/000137, with an internationalfiling date of Apr. 3, 2003 and claiming priority of India PatentApplication 169/MAS/2003 filed on Mar. 3, 2003, which documents are allincorporated herein by reference.

TECHNICAL FIELD

This invention relates to a product (nutritional supplement) to correctdyslipidemia, reduce inflammation and to reduce fasting sugar levels inblood in animals and humans and to combat intima-media thickening thusaffording significantly improved therapeutic and prophylacticcardioprotection, the said composition being an extract of Emblicaofficinalis, Gaertn (Euphorbiaceae) prepared without using any organicsolvent and without resorting to any chemical processing steps.

BACKGROUND OF THE INVENTION

E. officinalis (Amla) fruit is one of the key constituents of thecelebrated Ayurvedic preparation, Chyavanaprash, used in India forthousands of years as a vitalizing and rejuvenating health tonic. Thelow molecular weight hydrolyzable, gallo- and ellagi tannins (Ghosal, S.et al., Indian J. Chem., 1996, 35B, 941-48) of the fruit providemulti-pronged benefits arising out of their antioxidative,hypocholesterolemic, immunomodulating and HMG CoA reductase inhibitiveproperties. While its LDL-cholesterol lowering property has beendescribed in published literature, the more desirable property ofenhancing HDL cholesterol, as described in the present invention, wasnot noted before. Similarly, no earlier work had studied theantiinflammatory properties of amla, as observed in the presentinvention. Reduction of fasting glucose levels consequent to amlaconsumption is another desirable property observed in the presentinvention. We have also now surprisingly found that E. officinalis fruitextract reduces intima-media thickening in experimental animals. Thisobservation, though limited to experimental animals, is also reportedfor the first time. The cumulative effect of the reduction of multiplerisk factors by amla extract is the potential regression of this majordisease. Thus the beneficial effects of the present inventivecomposition goes beyond the simple correction of LDL cholesterol levels,as achieved in previous studies.

Coronary heart disease (CHD) continues to be the major cause ofpremature death in most developed and developing countries. A low levelof HDL cholesterol is the second most important risk factor for CHD, asdemonstrated in numerous clinical and epidemiological studies (Gorden,D. and Rifkind, H. M., N. Engl. J. Med., 1989, 321:1311-1315; Brewer,Jr., H. B., New Engl. J. Med., 2004, 350:1491-94) and HDL has emerged,during the past decade, as a new potential target for the treatment ofcardiovascular diseases. The key role of HDL as a carrier of excesscellular cholesterol in the reverse cholesterol transport pathway isbelieved to provide protection against atherosclerosis. In reversecholesterol transport, peripheral tissues, for example, vessel-wallmacrophages, remove their excess cholesterol through the ATP-bindingcassette transporter 1 (ABCA1) to poorly lipidated apolipoprotein A-I,forming pre-β-HDL. Lecithin-cholesterol acyltransferase then esterifiesfree cholesterol to cholesteryl esters, converting pre-β-HDL to maturespherical α-HDL.

HDL cholesterol is transported to the liver by two pathways: 1) it isdelivered directly to the liver through interaction with the scavengerreceptor, class B, type I (SR-BI); 2) cholesteryl esters in HDL aretransferred by the cholesterol ester transferase protein (CETP) tovery-low-density-lipoproteins (VLDL) and low-density lipoproteins (LDL)and are then returned to the liver through the LDL receptor. HDLcholesterol that is taken up by the liver is then excreted in the formof bile acids and cholesterol, completing the process of reversecholesterol transport (Brewer, H. B. Jr., Arterioscl. Thromb. Vasc.Biol., 2004, 24:387-91). HDL is believed to have the ability to removecholesterol from macrophages, thus preventing the formation of foamcells.

A second beneficial role of HDL in atherosclerosis is in protecting LDLfrom oxidation (Navab, M. et al, Circulation, 2002, 105:290-92). Unlikenormal LDL, oxidized LDL is readily taken up by macrophage scavengerreceptor SR-A or CD36 resulting in the formation of foam cells. Foamcells are a major component of the early atherosclerotic lesion.Further, HDL may slow the progression of lesions by selectivelydecreasing the production of endothelial cell-adhesion molecules thatfacilitate the uptake of cells into the vessel wall (Barter, P. J., etal, Curr. Opin. Lipid, 2002, 13:285-88). HDL may also prolong thehalf-life of prostacycline and preserve its vasodilatory effect(Mackness, M. I. et al, Atherosclerosis, 1993, 104:129-35).

Several lines of evidence support the concept that increasing the HDLlevel may provide protection against the development of atherosclerosis.Epidemiologic studies have shown an inverse relation between HDLcholesterol levels and the risk of cardiovascular disease. Increasingthe HDL cholesterol level by 1 mg may reduce the risk of cardiovasculardisease by 2 to 3 percent. Overexpressing the apo-A-I gene in transgenicmice and rabbits and infusing complexes consisting of apo A-I andphospholipids into hyperlipidemic rabbits increase HDL cholesterollevels and decrease the development of atherosclerosis (Brewer, H B,Jr., loc. cit). In humans, infusing either of these complexes orpro-apo-A-I results in short term increase in HDL cholesterol, biliarycholesterol and fecal cholesterol loss, reinforcing the concept thatelevating the HDL cholesterol level decreases the risk of cardiovasculardisease.

More than 40 percent of patients with myocardial infarction have lowHDL-C as a cardiac risk factor. (Genest, J. J., et al, Am. J. Cardiol.,1991, 67:1185-89). In the prospective and multicentric EuropeanConcerted Action on Thrombosis and Disabilities (ECAT) Angina PectorisStudy, Bolibar et al (Thromb. Haemost., 2000, 84:955-61) identified lowHDL-C and low apoA-I as the most important biochemical risk factors forcoronary events in patients with angiographically assessed CHD. Byconvention, the risk threshold value of HDL-C has been defined as 35mg/dL (0.9 mmol/L) in men and 45 mg/dL (1.15 mmol/L) in women [Expertpanel on detection, evaluation and treatment of high blood cholesterolin adults. The second report of the National Cholesterol EducationProgram (NCEP) expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults (Adult Treatment Panel II).Circulation. 1994; 89:1329-1445)]. Because of interaction, the strengthof the association between HDL-C and cardiovascular risk depends on thepresence of additional risk factors. Therefore, threshold values arehigher in men with diabetes mellitus or hypercholesterolemia or in thepresence of multiple risk factors (von Eckardstein A, and Assmann G.Curr Opin Lipidol. 2000; 11:627-637). Low HDL-C has been identified asthe most frequent familial dyslipoproteinemia in patients with prematuremyocardial infarction (Genest, J. J. Jr., Circulation. 1992;85:2025-2033). Finally, in the Helsinki Heart Study (Manninen, V. et al,Circulation. 1992; 85:37-45) and the High-Density-LipoproteinCholesterol Intervention Trial of the Department of Veterans Affairs(VA-HIT) study (Rubins, H. B. et al, N Engl J Med. 1999; 341:410-418),increases of HDL-C on treatment with gemfibrozil were correlated withthe prevention of CHD events. Thus, HDL-C has become an importantcomponent of algorithms to assess the global cardiovascular risk ofpatients and also a target for therapeutic intervention and for thedefinition of treatment goals.

Strategies to correct dyslipidemia in atherosclerosis generally involvediet and/or drugs. The threshold serum total cholesterol and LDLcholesterol concentrations above which diet and drug therapy should beinitiated, as well as the goals of therapy, have been defined by theNational Cholesterol Education Program (JAMA, 1993,269:3015-23). Thetarget serum LDL-C is <160 mg/dl (4.3 mmol/l) for patients with no riskfactors or only one risk factor for CHD; <130 mg/dl (3.4 mmol/l) forpatients with 2 or more risk factors and less than 100 mg/dl (2.6mmol/l) for those with CHD. Persons with diabetes also fall into thethird category. A reasonable target for triglyceride concentration is200 mg/dl or less; higher values are associated with a doubling of therisk of cardiovascular disease when serum cholesterol concentrationexceeds 240 mg/dl or when the LDL-C/HDL-C ratio exceeds 5:1.

A number of studies have shown that reducing serum LDL-C below thetarget levels does not necessarily result in proportional reduction inthe risk of CHD [(The Scandinavian Simvastatin Survival Study Group.Randomized trial of cholesterol lowering in 4444 patients with coronaryheart disease, Lancet, 1994, 344:1383-89; Shepherd, J. et al, N Engl. J.Med., 1995, 333:1301-7; Sachs, F. M. et al, N Engl. J. Med., 1998,315:1001-9; Circulation, 1998, 97:1446-52; The West of Scotland CoronaryPrevention Study Group, Circulation, 1998, 97:1440-45; Pederson, T. R.,Circulation, 1998, 97:1453-60] because of the attenuation of thecholesterol-heart disease relation at lower serum cholesterolconcentrations (Grundy, S. M., Circulation, 1998, 97:1436-39).

Dietary treatment of hyperlipidemia is a necessary foundation for drugtreatment. Depending on the degree of hyperlipidemia, the Step I andStep II diets can be introduced sequentially. The Step II diet containsno more than 30% of calories from fat, less than 7% of calories fromsaturated fatty acids and less than 200 mg of cholesterol per day. Inlong term studies, the Step II diet decreased serum LDL-C concentrations8-15% (Knopp, R. H., et al, JAMA, 1997, 278:1509-15; Walden, C. E.,Arterioscl. Thromb. Vasc. Biol., 1997, 17:375-82; Denke, M. A., Arch.Intern. Med., 1995, 156:17-26). Diets more restricted in fat than theStep II diet result in little additional reduction in LDL-C, raise serumTG concentration and lower HDL-C.

The point to note, from the above, is that reducing LDL-C alone is oflittle value in reducing the risk of CHD. Further, diets meant forreducing LDL-C may reduce HDL-C to a similar degree (Hunninghake, D. B.et al, N. Engl. J. Med., 1993, 328:1213-19; Schaefer, E. J., et al,Arterioscl. Thromb. Vasc. Biol., 1995, 15:1079-85); Stefanick, M. L., NEngl. J. Med., 1998, 339:12-20).

Drug therapy is resorted to when the desired effects are not achievedwith diets alone. Statins are the most popular among the lipid loweringdrugs. These drugs lower serum LDL-C concentrations by upregulatingLDL-receptor activity as well as reducing the entry of LDL into thecirculation. The maximal reductions achieved with a statin ranges from24-60%. Statins also reduce the serum TG levels; but they are ofteninsufficient. Statins are ineffective in the treatment of patients withchylomicronemia. Adverse effects of statins include, gastrointestinalupset, muscle aches and hepatitis. Rarer problems include myopathy(muscle pain with serum creatine kinase concentrations more than 1,000 Uper liter), rashes, peripheral neuropathy, insomnia, bad or vivid dreamsand difficulty in sleeping or concentrating (Abramowica, M., Med. Lett.,1996, 38:67-70; Vgontzas, A. N. et al, Clin. Pharmacol. Ther., 1991,50:730-37; Roth, T. et al, Clin. Cardiol., 1992, 15:426-32; Partinen, M.et al, Am. J. Cardiol., 1994, 73:876-80). Other lipid-lowering drugsinclude bile acid-binding resins (e.g, cholesteramine and colestipol),nicotinic acid, and fibrates.

Drug therapy is not recommended for premenopausal women and men under 35years of age unless they have serum LDL-C concentrations of more than220 mg/dl (5.7 mmol/l), because their immediate risk of heart disease islow [Summary of the second report of the National Cholesterol EducationProgram (NCEP): expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults, JAMA, 1993, 269:3015-23].

Thus, diets alone or in conjunction with lipid lowering drugs fail toyield the desired goal of safe lipid lowering. However, this goal isachievable with the present inventive composition containing the activeprinciples of Emblica officinalis. Emblica has been in safe use in Indiafor thousands of years as component of Ayurvedic preparations. Thecomposition offers the twin benefits of reducing the harmful LDLcholesterol and enhancing the desirable HDL cholesterol.

Further, the composition was found to reduce the intima-media thickeningof the arteries in experimental animals which is an added benefit. Suchan effect has not been observed before.

Amla's cholesterol lowering effects have been reported in a few studies.Thakur et al studied the effect of amla on cholesterol-inducedatherosclerosis in rabbits (Thakur, C. P. and Mandal, K., Indian J MedRes, 79: 142-6 1984; Thakur C P, et al. Int J Cardiol, 1988, 21:167-75).The control group was fed with cholesterol alone and the experimentalgroup, amla and cholesterol for 16 weeks. Cholesterolemia was found tobe significantly less in amla group (205 mg/dl) than in the controlgroup (630 mg/dl). Aortic sudanophilia was significantly less in theamla group (12%) than in the control group (38%). The cholesterolcontents of the liver and aorta, respectively, were significantly lessin the amla group (46 mg/100 g, 42 mg/100 g), than in the control group(604 mg/100 g, 116 mg/100 g). Amla did not influence serum triglyceride(TG) levels, euglobulin clot lysis time or platelet adhesiveness.Another study (Mather R, et al. J Ethnopharmacol, 1996, 50:61-68) foundthat serum cholesterol, TG, phospholipids and LDL cholesterol werelowered 82%, 66%, 77% and 90%, respectively, when fresh amla juice wasfed to rabbits. Aortic plaques were regressed. Amla juice-treatedrabbits excreted more cholesterol and phospholipids. Similar resultshave been reported by others (Mishra M, et al. Br J Exp Pathol, 1981,62:526-28; Tariq M, et al. Indian J Exp Biol, 1977, 15:485-86).

Amla was found to inhibit cholesterol synthesis in rats (Anila I,Vijayalakshmi N R, J Ethnopharmacol, 2002, 79:81-87) by inhibiting thecholesterol synthesizing enzyme HMG CoA reductase. Degradation andelimination of cholesterol was noted, and thus the hypercholesterolemiainduced by amla was suggested to be due to inhibition of synthesis andenhancement of degradation.

Oxidized LDL (ox-LDL) is one of the etiological factors ofatherogenesis. A study was conducted to see if the antiatherogeniceffects of amla was due to its effect on ox-LDL (Duan W, et al.Yakagaku, 2005, 125:587-91). Human umbilical vein endothelial cells(HUVEC) was incubated with ox-LDL and corilagin and its analogue Dgg 16(present in amla) and then incubated with monocytes. Malondialdehyde(MDA) in the culture media was then determined. Monocytes adhering tothe HUVEC were counted by cytometry. In another experiment, rat vascularsmooth muscle cells (VSMC) were incubated in the media with or withoutox-LDL and with corilagin and Dgg 16 at different doses and cellproliferation was assayed. Both corilagin and Dgg 16 were able to reduceMDA, prevented HUVEC from adhering to monocytes, and inhibited VSMCproliferation induced by ox-LDL. The authors concluded that theantiatherogenic effect of amla is due to corilagin and Dgg-16. Ethylacetate extract of amla was a stronger antioxidant than probucol inpreventing LDL oxidation (Kim H J, et al. J Nutr Sci Vitaminol, 2005,51:1812-18).

So far no study has reported the HDL enhancing effect of amla, asdescribed in the present invention.

Inflammation

An important component of atherosclerotic process is inflammation (Ross,R, N Engl J Med, 1999, 340:115-26; Libby P, Nature, 2002, 420:868-74).The fundamental appreciation that inflammation is an important andpossibly even obligatory component of lesion initiation and progression,and also participates in the plaque rupture that mediates thromboticcomplications and clinical events, has fundamentally changed the view ofthe pathogenesis of atherosclerosis. Thus correcting dyslipidemia alonedoes not reduce the risk of cardiovascular disease, or of clinicalevents in patients with established disease.

Considering the importance of inflammation in atherosclerosis, attentionhas been directed to search for mediators which are appropriate formonitoring inflammation. The most reliable marker for inflammation hasbeen found to be the blood levels of C-reactive protein (CRP) (Pepys MB, Hirschfield, G M, J Clin Invest, 2003, 111:1805-12; Ridker P M, etal. N Engl J Med, 2005, 352:20-28). Clinical studies have shown theassociation of elevated plasma levels of CRP and increasedcardiovascular risk (Shishehbor M H, et al. Cleve Clin J Med, 2003,70:634-40). Especially chronically elevated CRP levels measured by highsensitive assays (hs-CRP) can independently predict the risk ofcardiovascular events (Ridker P M, Circulation, 2003, 107:363-69).Patients with acute coronary syndrome often have elevated plasma levelsof CRP (Nieminem M S, et al. Eur Heart J, 1993, 14:(suppl K):12-16). CRPhas also been reported to determine the prognosis of developing arterialischemia in healthy persons (Ridker P M, et al, N Engl J Med, 2000,342:836-42; Harris T B, et al. Am J Med, 1999, 106:506-12; Ridker P m etal, JAMA, 2001, 285:2481-85). In patients with cardiovascular diseasewho underwent coronary intervention, it can predict the risk ofdeveloping myocardial infarction and death (Chew D P et al, Circulation,2001, 104:992-97; Lenderink T, et al. Eur Heart J, 2003, 24:77-86). CRPis a sensitive marker for inflammation leading to arteriosclerosis andmonitors the inflammatory process in the arterial wall. It also has adirect influence on arterial injury. In the presence of CRP, endothelialadhesion molecules are significantly upregulated (Paceri V, et al.Circulation, 2000, 102:2165-68). CRP furthermore can stimulate monocytesto produce tissue factor, an important initiator of the clotting cascade(Cermal J, et al. Blood, 1993, 82:513-20). These data underlie theutility of CRP measurements in predisposed persons, and also suggestrole for antiinflammatory therapy in those patients. It has beensuggested that patients with coronary artery disease do benefit fromreduction of CRP levels (Tomoda H, Akoi N, Am Heart J, 2000, 140:324-28;Ridker P M et al. Circulation, 1999, 100:230-35; Ridker P M, et al. NEngl J Med, 2005, 352:20-28).

CRP, thus, is not just a marker of inflammation, but an agent involvedin the atherogenic process as well as a predictor of future cardiacevents. Hence the need to keep the CRP to normal levels.

Amla's effect on CRP and on inflammation in general, has not beenreported earlier. In one embodiment, the disclosed amla product has beenfound to be a robust agent to reduce CRP in human volunteers in thepresent invention.

Hyperglycemia

Diabetes mellitus magnifies the risk of cardiovascular morbidity andmortality (Resnick He, et al. J Clin Epidemiol, 2001, 54:869-76; BeckmanJ A, et al. JAMA, 2002, 287:2570-81). Besides the well-recognizedmicrovascular complications of diabetes such as nephropathy andretinopathy, there is a growing epidemic of macrovascular complicationsincluding diseases of the coronary arteries, peripheral arteries andcarotid vessels, particularly in the burgeoning type 2 diabeticpopulation.

Coronary artery disease (CAD) causes much of the serious morbidity andmortality in diabetic patients who have a 2- to 4-fold increase in riskof CAD (Haffner S M, et al. N Engl J Med, 1998, 339:229-234). This hasbeen observed a number of large trials (Kjaergaard Sc, et al, Scand JCardiovasc J, 1999, 33:166-70; Malmberg K, et al, Circulation, 2002,102:1014-19; Zuanetti G, et al. J Am Coll Cardiol, 1993, 22:1788-94;Shindler D M et al, J Am Coll Cardiol, 2000, 36:1097-1103). Patientswith diabetes also have an adverse long-term prognosis after myocardialinfarction (MI), including increased rates of reinfarction, congestiveheart failure and death (Malmberg K, et al, Circulation, 2002,102:1014-19). A Finnish study on trends of MI showed that diabetesincreased 28-day mortality by 58% in men and 160% in women (Miettinen H,et al., Diabetes Care, 1998, 21:69-75). The 5-year mortality ratefollowing MI may be as high as 50% for diabetic patients, more thandouble that of nondiabetic patients (Herlitz J, et al. Diabetes Med,1998, 15:308-14). Such results led the Adult Treatment Panel III of theNational Cholesterol Education Program to establish diabetes as a CADrisk equivalent mandating aggressive antiatherosclerotic therapy (JAMA,2001, 285:2486-97).

Hyperglycemia (increased blood sugar levels), a cardinal manifestationof diabetes, adversely affects vascular functions, lipids, andcoagulation. Intensive treatment of hyperglycemia reduces the risk ofmicrovascular complications such as nephropathy and retinopathy, asshown by the United Kingdom Prospective Diabetes Study (UKPDS) (UKPDS33, Lancet, 1998, 352:837-53). In a meta-analysis of more than 95,000diabetic patients, increases in cardiovascular risk depended directly onplasma glucose concentrations and began with concentrations below thediabetic threshold (Coutinho M, et al. Diabetes Care, 1999, 22:233-40).

Diabetes also causes abnormalities in lipid profile, including elevatedtriglyceride levels, decreased HDL levels and increased levels of small,dense LDL. Elevated levels of triglyceride-rich lipoproteins lower HDLlevels by promoting exchanges of cholesterol from HDL to VLDL (SnidermanA D, et al. Ann Intern Med, 2001, 135:447-59). Diabetic patients withCAD more commonly have elevated triglyceride and low HDL levels thanelevated total cholesterol and LDL cholesterol levels (Rubins H B, etal. Am J Cardiol, 1995, 75:1196-1201). HDL normally protects LDL fromoxidation, but this ability is impaired in diabetic patients (Gowri M S,et al. Arterioscl Thromb Vasc Biol, 1999, 19:2226-33).

Thus controlling hyperglycemia is important to prevent diabetic as wellas cardiovascular complications.

Hypoglycemic effects of amla has not been described earlier. However,such effects of two polyherbal compositions (Triphala and Hyponidd) ofwhich amla is a constituent have been reported (Sabu M C, Kuttan R, JEthnopharmacol, 2002, 81:155-60; Babu P S et al. J Pharm Pharmacol,2004, 56:1435-42). Triphala is a mixture of three herbal extracts,whereas ten herbs constitute Hyponidd. The latter also contain knownhypoglycemic herbs such as Momordica charantia and Gymnema sylvestre. Asdescribed in the present invention, the amla product is found to possesshypoglycemic properties.

Thyroid Dysfunction

Thyroid hormone excess and deficiency are common (Hollowell J G, et al.J Clin Endocrinol Metab, 2002, 87:489-99; Vanderpump M P, et al. ClinEndocrinol (Oxford), 1995, 43:55-68) and are readily diagnosed andtreated. A number of studies suggest that abnormal levels of thyroidstimulating hormone (TSH) may represent a novel risk factor forcardiovascular diseases (Hak A E, et al. Ann Intern Med, 2000,132:270-78; Parle T V, et al. Lancet, 2001, 358:861-65; Imaizumi, M, etal. J Clin Endocrinol Metab, 2004, 89:3365-70; Kvetny J, et al. ClinEndocrinol (Oxford), 2004, 61:232-38; Walsh J P, et al. Arch Intern Med,2005, 165:2467-72). Even mildly altered thyroid status reportedlyaffects serum cholesterol levels (Danese M D, et al. J Clin EndocrinolMetab, 2000, 85:2993-3001; Vierhapper H, et al Thyroid, 200, 10:981-84;Canaris G J, et al. Arch Intern Med, 2000, 150:526-34) heart rhythm(Sawin C T, et al. N Engl J Med, 1994, 331:1249-52) and rate (Bell G M,et al. Clin Endocrinol (Oxford), 1983, 18:511-16), ventricular function(Biondi B, et al. J Clin Endocrinol Metab, 2000, 85:4701-05; Idem, Ibid,1999, 84:2064-67), risk of coronary artery disease (Hak A E, Loc cit;Walsh J P, loc cit; Cappola A R, et al. J Clin Endocrinol Metab, 2003,88:2438-44).

Thyroidisms are classified into various categories (Cappola A R, et al.JAMA, 2006, 295:1033-41) as euthyroidism (normal TSH concentrations(0.45 to 4.5 mU/L), subclinical hyperthyroidism (TSH concentration 0.10to 0.44 mU/L0, or less than 0.10 mU/L with a normal free thyroxine (FT4)concentration; subclinical hypothyroidism (TSH concentration more than4.5 mU/L and less than 20 mU/L with a normal FT4 concentration, andovert hypothyroidism with a TSH concentration of 20 mU/L or more. Inovert hyperthyroidism, the TSH levels are suppressed much below thenormal levels, usually undetectable, or can be measured in athird-generation assay capable of detecting 0.01 mU/L (Shrier M D,Burman K D, Am. Fam Phys, 2002, 65:431-38).

Thyroid hormone has relevant effects on the cardiovascular system (KleinI, Ojama K, N Engl J Med, 2001, 344:501-09; Fazio, S et al. Recent ProgHorm Res, 2004, 59:31-50). Many symptoms and signs recognized inpatients with overt hyper- and hypothyroidisms are due to the increasedor reduced action of the thyroid hormone on the heart and vascularsystem, respectively Subclinical thyroid dysfunction may affect thecardiovascular system, which may increase the cardiovascular risk. Inaddition, patients with acute or cardiovascular disorders haveabnormalities in peripheral thyroid hormone metabolism that may altercardiac functions. The morbidity and mortality associated withhypothyroidism are apparently related to the atherogenic andprothrombotic vascular modifications that follow thyroid hormonedeficiency, whereas heart failure and particularly atrial fibrillationand its thromboembolic complications are the primary consequences ofhyperthyroidism. In both cases, return to normal thyroid levels correctsthe cardiac abnormalities caused by thyroid dysfunction.

Amla has been found to be beneficial in hyperthyroidism, though thestudy was limited to mice (Panda S, Kar A, Pharmazie, 2003, 58:753-56).Amla fruit extract was evaluated for its effects on the L-thyroxine(L-T4)-induced hyperthyroidism in mice. While an increase in serum T3(triiodothyronine) and T4 (thyroxine) concentrations, and in a thyroiddependent parameter, hepatic glucose 6-phospatase (glu-6-pase) activitywas observed in L-T4 (0.5 mg/kg/d) treated animals, simultaneous oraladministration of the plant extract at a dose of 250 mg/kg/d (p.o.) for30 days in hyperthyroid mice reduced T3 and T4 concentrations by 64 and70% respectively as compared to a standard antithyroid drug, propylthiouracil that decreased the levels of the thyroid hormones by 59 and40% respectively. The plant extract also maintained nearly normal valueof glu-6-pase activity in hyperthyroid mice.

Results reported in the present invention is the first report on theeffect of amla in thyroid dysfunction in humans.

Intima-Media Thickening

The increased thickness of intima plus media of the carotid artery isassociated with the prevalence of cardiovascular diseases and a numberof studies have shown a positive association between cardiovascular riskfactors and carotid intima-media thickness (IMT)(O'Leary, D. H. et al.,Stroke, 1992, 22:1156-63; 1992, 23:1752-60; New Engl. J. Med., 1999,340(1):14-22; Howard, N. et al, Ann. Int. Med., 1998, 128(4):262-69);Zureik, M. et al, Stroke, 1999, 30:550-55; del Sol, A. I. et al, Stroke,2001, 32:1532-38). Howard et al (loc.cit.) makes the followingstatement: For each 0.03 mm increase per year in carotid arterial IMT,the relative risk for non-fatal myocardial infarction or coronary deathwas 2.2 (95% CI, 1.4-3.6) and the relative risk for any coronary eventwas 3.1. Absolute IMT was also related to risk for clinical coronaryevents. Absolute thickness and progression in thickness predicted riskfor coronary events beyond that predicted by coronary arterial measuresof atherosclerosis and lipid measurements. A growing number ofepidemiological studies and clinical trials use IMT as an early markerof systemic atherosclerosis (Zanchetti, A., et al, J. Hypertens., 1998,16:949-61; MacMahon, S. et al, Circulation, 1998, 97:178-90: Borhani, N.O. et al, JAMA, 1996, 124:548-56; Hodis, H. N. et al, Ann. Intern. Med.,1996, 124:548-52).

IMT is increasingly being used in clinical trials as surrogate end pointfor determining the success of interventions that lower risk factors foratherosclerosis. To distinguish early atherosclerotic plaque formationfrom thickening of the intima-media, the following consensus has evolved(Touboul, P. J. et al, Mannheim Intima-Media Thickness Consensus. onBehalf of the Advisory Board of the 3rd Watching the Risk Symposium2004, 13th European Stroke Conference, Mannheim, Germany, May 14, 2004,Cerebrovasc. Dis., 2004, 18(4):346-49): Plaque is defined as a focalstructure that encroaches into the arterial lumen of at least 0.5 mm or50% of the surrounding IMT value or demonstrates a thickness of ≧1.5 mmas measured from the media-adventitia interface to the intima-lumeninterface. Standard use of IMT measurements is recommended in allepidemiological and interventional trials dealing with vascular diseasesto improve characterization of the population investigated.

Endothelial vasodilator dysfunction and carotid IMT are two indicatorsof subclinical cardiovascular disease. In a study of a large,community-based cohort of young adults (aged 24-39 years), Jounala et al(Circulation, 2004, 110(18):2918-23) found that IMT was inverselyassociated with endothelium-dependent brachial artery flow-mediateddilation (FMD). The number of risk factors was correlated with increasedIMT in subjects with evidence of endothelial dysfunction. In a relatedstudy, FMD and glyceryl trinitrate-induced endothelium-independentvasodilation (GTN) were measured in the brachial artery. IMT of thecommon carotid artery and insulin sensitivity were also measured. Therewas a significant positive relation between insulin resistance, asmeasured by steady-state glucose levels, and IMT. Insulin resistance wasnegatively correlated with both FMD and GTN. This indicates that bothFMD and GTN were also negatively correlated with IMT (Suzuki, M. et al.,Am. J. Hypertens, 2004, 17(3):228-32). Similarly, in a study on 252healthy adults, IMT was significantly greater in subjects withsubclinical aortic valve sclerosis (Yamamura, Y., et al, Am. J.Cardiol., 2004, 94(6):837-39). To determine whether IMT is related to anincreased risk of cardiovascular event after percutaneous coronaryangioplasty (PTCA), IMT was measured within 2 days following PTCA in 88patients (mean age 62 years) in another study. A common carotid IMT >0.7mm was associated with an increased risk of cardiac events after PTCA(Lacroix, P. et al., Int. Angiol., 2003, 22(3):279-83).

Low HDL cholesterol was associated with increased IMT independent ofother risk factors in healthy subjects from families with low HDLcholesterol (Alagona, C., et al, Eur. J. Clin. Invest., 2003,33(6):457-63). Conversely, increased HDL cholesterol was negativelycorrelated with IMT (Blankenhorn, D. H., et al, Circulation, 1993,88(1)20-28; Bonithon-Kopp, C. et al, Arterioscl. Thromb. Vasc. Biol,1996, 16:310-16).

A statistically significant IMT greater than 0.8 mm was associated withcoronary artery disease with an odds ratio of 2.4 in Indian subjects(Jadhav, U. M. and Kadam, N. N., Indian Heart. J., 2001, 53(4):458-62).The same authors also found (J. Assoc. Physicians India, 2002,50:1124-29) a statistically significant association of microalbuminuriawith IMT and coronary artery disease in diabetic patients.

In a large population-based of 6943 subjects, carotid IMT and aorticcalcification were found to be the strongest predictors of stroke(Hollander, M. et al., Stroke, 2003, (10):2368-72).

From the foregoing, the importance of IMT in cardiovascular diseasemanagement is amply evident. Fortunately, IMT is modifiable. Varioussynthetic drugs, for example, colestipol plus niacin (Blankenhorn, D.H., Circulation, 1993, 88(1):20-28), candesartan (Igarashi, M. et al,Hypertension, 2001, 38(6):1255-59), simvastatin (Detmers, P. A., et al,Circulation, 2002, 106(1):20-23), rampamycin with tacrolimus orcyclosporin (Weller, J. R., et al, Br. J. Surg., 2002, 89(11):1390-95),calcium channel blockers (Wang, J. G. and Staessen, J. A., J. Am. Soc.Nephrol., 2002, 13 Suppl:S208-15), sulfated oligosaccharide PI-88(Francis, D. J., et al, Circ. Res., 2003, 92(18):70-77), fluvastatin(Ye, P. et al, Chin. Med. Sci. J., 2000, 15(3):140-44), lovastatin(Furberg, C. D., et al, Circulation, 1994, 90:1679-87), chemicallymodified tetracycline (Islam, M. M., et al, Am. J. Pathol., 2003,163(4):1557-66) reduce IMT. There are very few natural products whichare reported to suppress intimal thickening. Thus, the finding thatEmblica extract can reduce IMT assumes great significance.

The disclosed amla product was found to reduce IMT in rabbits. Thoughthe reduction in IMT has not been proven in humans, it is expected suchresults may be achieved in human beings as well because IMT is inverselyassociated with HDL concentration (Watanabe et al. Arterioscl Thrombvasc Biol, 2006, 26:897-902; Alagona C et al. Atherosclerosis, 2002,165:309-16; Eur J Clin Nutr, 2003, 33:457-63). IMT is also positivelycorrelated with CRP (Wang T S et al. Arterioscl Thromb Vasc Biol, 2002,22:1662-67; Sitzer M, et al. J Cardiovasc Risk, 2002, 9:97-103).Diabetes is also associated with increased IMT (Mohan V, et al. DiabetesMed, 2006, 23:845-50; Wagenknecht L E et al. Diabetes Care, 1998, 21:1812-18; Brohall G, et al. Diabet Med, 2006, 23:609-16). Thus, it isreasonable to expect that the disclosed amla product would reduce IMT,indirectly indicating regression of atherosclerosis.

The present inventive composition, by its direct effects ondyslipidemia, inflammation, hyperglycemia and intima-media thickening,thus offers much improved cardioprotection than any other similarproduct, natural or synthetic, with the added benefit of its time-testedsafety.

Ghosal has disclosed a process for preparation of an extract of Emblicaofficinalis (U.S. Pat. No. 6,124,268) and a few more by the sameinventor for various applications of the preparation, such as,stabilization of vitamin C (U.S. Pat. No. 6,235,721), inhibitingplatelet aggregation (U.S. Pat. No. 6,290,996) and antioxidant to blockfree radical process (U.S. Pat. No. 6,362,167). In none of the abovepatents, the hypocholesterolemic action, and more specifically, its HDLenhancing property or other properties described in the presentinvention, have been described. The present inventive preparation alsomaterially differs in composition from that described by Ghosal. Theextraction process described by Ghosal involves treating the fruit pulpwith water containing 1% sodium chloride which was then left at roomtemperature for 12 hours followed by keeping the mixture at 10° C. for 3days and thus is very time-consuming, costly and tedious. Further, heuses sodium chloride solution for extraction and apparently, the saltremains in the final preparation. This may not be desirable, given theadverse affects of salts for patients with hypertension which is closelyassociated with cardiac diseases. Further, going by the examples givenin the said Ghosal patent, the content of active principles, which hecalls as the antioxidant fraction, is less than 4% in the finalpreparation. Thus, a more commercially attractive process would behighly desirable.

BRIEF DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides for a preparation an extractof amla, with standardized contents of bioactive principles suitable fortherapeutic as well as prophylactic treatment of dyslipidemia, reductionof inflammation and cardiovascular events by lowering CRP levels,reduction of fasting blood sugar levels thus lowering diabetic andcardiovascular complications induced by hyperglycemia and correctthyroid dysfunction. The disclosed Amla product thus affords completeprotection against the major risk factors of cardiovascular diseases.This is further demonstrated by the reduction in intima-media thicknessof arteries indicating the power of the disclosed amla product inreversing the atherosclerotic process. Reduction of the harmful LDLcholesterol, and more importantly, the enhancement of the beneficial HDLcholesterol was observed. Though this has not been proven in humans, itis expected such results may be achieved in human beings as well becauseIMT is inversely associated with HDL concentration and any interventionto increase serum HDL-C level would reduce IMT.

One objective of the present invention is to provide a safe naturalproduct for treatment of lipid disorders connected with coronary heartdisease and stroke without the disadvantages associated with the use ofsynthetic lipid lowering agents such as statins. More specifically, theinvention aims at providing a composition which lowers the harmful totalcholesterol, LDL cholesterol and triglycerides, and at the same timeincreasing the beneficial HDL cholesterol contents. Low HDL cholesterolis considered as the second most important predictor of coronary heartdisease (CHD).

Another objective of the present invention is to provide for a safe,natural product composition to reduce plasma CRP levels and inflammationassociated with atherosclerosis, thus reducing risk for future clinicalevents such as myocardial infarction and stroke.

Another objective of the present invention is to provide for a safe,natural product composition to reduce hyperglycemia in diabetic andpre-diabetic patients, who are at increased risk of coronary arterydiseases as well as reduce the risk of microvascular complicationsassociated with diabetes such as nephropathy and retinopathy.

Another objective of the present invention is to provide for a safe,natural product composition to potentially reduce the intima-mediathickness of the arteries. Another objective of the invention is toprovide for a composition for potential reduction of intima-mediathickening observed in heart patients and which is considered as animportant marker and predictor of CHD.

Another objective of the present invention is to provide a safe, naturalproduct composition to correct thyroid dysfunction, which is nowrecognized as a risk factor for cardiovascular diseases.

The present invention thus aims at providing a safe natural productcomposition to reduce the risk factors associated with coronary arterydiseases and induce remission.

A further objective of the present invention is to provide an easy andeconomical process for the commercial preparation of E. officinalisextract offering the benefits described above.

A further objective of the invention is to provide a method of producinga product to correct hypercholesterolemia in a human including:

pulping fruits of Emblica Officinalis with demineralized water to createa slurry;

treating the slurry with pectinase to form a pectinase-treated slurry;

filtering the pectinase-treated slurry to create a solution; and

concentrating the solution to generate the product.

These and further objectives of the invention will be apparent from thedetailed description of the invention given below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Table 7, which provides the effect of product onhematological parameters in human patients; and

FIG. 2 shows Table 8, which provides effect of product on lipid profilein hypercholesterolemic patients.

DETAILED DESCRIPTION OF THE INVENTION

The three important components of coronary artery diseases are lipids,inflammation and immunity (for example, see Binder C J, et al. NatureMed, 2002, 8:1218-26). What initiates the atherosclerotic process is notentirely clear, and there are probably several different pathogenicprocesses that can elicit localized inflammatory responses in theartery. One prime candidate is minimally oxidized LDL, and late forms ofoxidized LDL (ox-LDL). Once trapped in the artery wall by binding toextracellular proteoglycans, a key event in atherogenesis (Williams K Jand Tobas I, Curr Opin Lipidol, 1998, 9:471-74; Skalen K, et al. Nature,2002, 417:750-54), LDL is oxidized by mechanisms still unknown orundergoes other types of modifications, such as non-enzymatic glycation,enzymatic degradation, aggregation, or a combination of these, all ofwhich results in alteration of the ‘self’. These modifications makes themodified LDL and leads to both cellular and humoral responses (Horkko,S, et al. Free Radic Biol Med, 2000, 28:1771-79. In addition, theoxidation of LDL generates oxidized lipids that are toxic,proinflammatory and pro-atherogenic (Pratico D, Trends Cardiovasc Med,2001, 11:112-16). Oxidized phospholipids can induce artery wall cells tosecrete chemotactic molecules (chemokines), activate endothelial cellsto express adhesion molecules, and induce expression of growth factorsthat facilitate the transformation of monocytes to macrophages andstimulate the proliferation of smooth muscle cells (Berliner J A, et al.Trends Cardiovasc Med, 2001, 11:112-16; Marathe G K, et al. TrendsCardiovasc Med, 2001, 11:139-42). Macrophages, a central mediator ininnate and adaptive immunity, are essential in lesion initiation andprogression (Glass C K and Witztum J L, Cell, 2001, 104:503-16; HanssonG K, et al. Circ Res, 2002, 91:281-91). Once activated they initiateoxidation of LDL and rapidly take up oxLDL through specific scavengerreceptors leading to foam cell formation (Witztum J l, et al. trendsCardiovasc Med, 2001, 11:93-102). This is a key event in diseaseprogression. Activated macrophages also secrete a variety ofpro-inflammatory molecules that affect lesion progression and plaquestability.

The present invention provides for a natural product that has been foundto directly influence two of the three components, namely lipid disorderand inflammation, of coronary artery diseases discussed above, withpotential for modulating the third component, namely immunity, as well.It would be difficult to assess the involvement of amla in modulatingthe immune system component, but earlier studies have clearly shown itsimmunomodulating properties (Nemmani K V et al. Indian J Exp Biol, 2002,40:282-87; Muruganandam A V, et al. Indian J Exp Biol, 2002, 40:1150-60;Bhattacharya A, et al. Indian J Exp Biol, 2002, 40:1161-63; Sai Ram. M,et al. J Ethnopharmacol, 2002, 81:5-10; Phytother Res, 2003, 17:430-33;Bhattacharya S K et al. Indian J Exp Biol, 2000, 38:945-47).

Thus, one embodiment of the disclosed amla product modulates all theimportant components of coronary artery diseases to induce regression ofthe disease. Further, one embodiment of the disclosed amla productreduces the fasting blood sugar levels, a confounding factor inatherosclerosis. The potential of the disclosed amla product in inducingregression is shown by the reduction in intima-media thickness inexperimental animals.

The present invention provides for a safe natural product for treatmentof disorders connected with coronary artery diseases, such as coronaryheart disease, stroke and peripheral artery disease, without thedisadvantages associated with the use of synthetic lipid lowering agentssuch as statins.

More specifically, the invention aims at providing a composition whichlowers the harmful LDL cholesterol and triglycerides and at the sametime increasing the beneficial HDL cholesterol contents. Low HDLcholesterol is considered as the second most important predictor ofcoronary heart disease (CHD) risk. One embodiment of the disclosed amlaproduct thus corrects dyslipidemia associated with coronary arterydiseases.

One embodiment of the disclosed amla product further reducesinflammation which is an integral part of coronary artery diseases, asevidenced by reduction in CRP levels. Since CRP is a predictor of futurecoronary events, reduction in CRP by the disclosed amla productindicates that the amla product directly affords reduction in risks ofclinical events such as myocardial infarction and stroke.

Furthermore, one embodiment of the disclosed amla product reduces theblood sugar levels in diabetics who are at an elevated risk of heartdiseases and corrects dyslipidemia associated with diabetes.

One embodiment of the amla product was also found to correct thyroiddysfunction (both hypo- and hyperthyroidisms) which has a direct effecton heart and vascular systems, and now recognized as a risk factor forcardiovascular diseases.

Further, though limited to animal studies, the composition was found toprevent smooth muscle cell proliferation and to reduce the intima-mediathickening associated with the process.

The present invention provides for a safe natural product for treatmentof lipid disorders connected with coronary heart disease and strokewithout the disadvantages associated with the use of synthetic lipidlowering agents such as statins. More specifically, the invention aimsat providing a composition which lowers the harmful LDL cholesterol andtriglycerides and at the same time increasing the beneficial HDLcholesterol contents. Low HDL cholesterol is considered as the secondmost important predictor of coronary heart disease (CHD) risk. Further,though limited to animal studies, the composition was found to preventsmooth muscle cell proliferation and to reduce the intima-mediathickening associated with the process.

The present composition uses the extract of fresh fruits of Emblicaofficinalis, a tree that occupies a prime position in Ayurvedicpreparations for its rejuvenating, vitalizing properties and above allits time-tested safety record. Amla is known as an immunomodulator andthis property also is believed to be acting in concert with the observedeffects to further induce remission of the disease. The extract isprocessed as detailed below to contain a minimum amount of activeconstituents which are believed to be the low molecular weighthydrolyzable tannins, especially, emblicanin A.

In one embodiment of the process, the invention provides for acomposition to correct lipid disorders associated with coronary heartdisease, namely high LDL cholesterol and triglycerides and low HDLcholesterol in blood.

In another embodiment, the invention provides for a composition toreduce inflammation associated with coronary artery diseases, asevidenced by the reduction in blood CRP levels.

In a further embodiment, the invention provides for a composition toreduce blood sugar levels which has a confounding effect on heartdiseases.

In another embodiment, the invention provides for a composition tocorrect thyroid dysfunctions.

In another embodiment of the invention a composition is provided forreducing the major risk factors associated with coronary artery diseasesand induce remission of the disease as evidenced by reduction in theintima-media thickness which is increasingly being used as a marker forheart diseases. Very few natural products have been reported to possessthis capability.

In yet another embodiment, the invention provides for a process for thecommercial scale preparation of such a composition without the use ofany organic solvent and without any chemical treatment or additives andthus possesses much superior properties than any of the lipid loweringagents known.

Accordingly, the pulp obtained from fresh amla fruits was treated with apectinase enzyme for a sufficient length of time at room temperature.The slurry was filtered to yield a clear filtrate which was thenspray-dried to get the composition as a dry, free-flowing powder.

The product was then encapsulated in hard gelatin capsules to contain500 mg of the spray-dried powder which supplies a minimum of 10 wt % ofemblicanin A.

In one embodiment, the invention provides a product having an extract offruits of Emblica Officinalis, wherein the extract is prepared withoutusing any organic solvent and without subjecting to any chemicaltreatment at any stage.

In one embodiment, the invention provides a method of producing aproduct including:

pulping fruits of Emblica Officinalis with demineralized water to createa slurry;

treating the slurry with pectinase to form a pectinase-treated slurry;

filtering the pectinase-treated slurry to create a solution; and

concentrating the solution to generate the product.

In one embodiment, the invention provides a method of producing aproduct to correct hypercholesterolemia in a human.

Acute and sub-acute toxicities of the products were tested in mice andrats, respectively. Up to a dosage level of 10 g/kg body weight producedno adverse effects such as increased motor activity, tremors, clonicconvulsions, piloerection, muscle spasm, hyperesthesia, ataxia,sedation, hypnosis and analgesia in mice. No mortality was recorded in72 hours. Sub-acute toxicity studies at a dosage level of 2 g/kg bodyweight for 3 months produced no toxic effects in rats.

The hypocholesterolemic properties of the composition were tested inrabbits. Rabbits were made hypercholesterolemic by oral feeding ofcholesterol for 4 months. At the end of 4 months, the treatment groupswere administered with the inventive composition for an additional 4months. Body weight measurements, haematological parameters and lipidprofiles of the animals were determined at regular intervals. Anear-reversal of the hypercholesterolemic conditions were observed inthese animals. There was also reduced activity of thecholesterol-synthesizing enzyme HMG CoA reductase and surprisingly, thethickness of intima plus media also were reduced to normal levels.

The lipid lowering properties of the composition were further tested inhypercholesterolemic human volunteers. The results were in generalagreement with those observed in the animal studies. More importantly,there was significant increase in the beneficial HDL cholesterol levels.Recent research has indicated that increasing the HDL cholesterol levelis even more important than reducing the LDL cholesterol level.

The composition was further tested in apparently healthy humanvolunteers, for its effect on blood CRP levels and fasting blood sugarlevels, in addition to its effect on the lipid profiles. The compositionshowed positive benefits in these aspects as well. Blood CRP level is amarker for systemic inflammation and a predictor of future cardiacevents. Blood sugar has a confounding effect on the disease.

The product can be administered to a human for a method of reducingserum cholesterol levels. The product can be administered to a human fora method of reducing at least one of serum LDL and VLDL cholesterolconcentrations. The product can be administered to a human for a methodof enhancing HDL cholesterol levels. The product can be administered toa human for a method of reducing triglyceride to correct dyslipedemia.The product can be administered to a human for a method of preventingsmooth muscle cell proliferation and reducing intima media thickening.The product can be administered to a human for a method of reducing HMGCoA reductase activity.

The product can be administered to a human for a method of correctingdyslipedemia. The product can be administered to a human for a method ofreducing inflammation. The product can be administered to a human for amethod to reduce fasting sugar levels in the blood. The product can beadministered to a human for a method of therapeutic and prophylacticcardioprotection. The product can be administered to an atherosclerotichuman patient for a method to induce regression of the atheroscleroticprocess, whereby dyslipedemia, inflammation and blood sugar levels arealso corrected. The product can be administered to a human patienthaving coronary artery diseases to induce regression of atheroscleroticprocess. The product can be administered to a patient having coronaryartery diseases to correct lipid abnormalities. The product can beadministered to a patient having coronary artery diseases to reduceserum total cholesterol. The product can be administered to a patienthaving coronary artery diseases to reduce VLDL cholesterol. The productcan be administered to a patient having coronary artery diseases toreduce LDL cholesterol. The product can be administered to a patienthaving coronary artery diseases to reduce triglyceride concentration.The product can be administered to a patient having coronary arterydiseases to elevate beneficial HDL cholesterol. The product can beadministered to a patient having coronary artery diseases to reduceinflammation associated with coronary artery disease. The product can beadministered to a patient having coronary artery disease to reduceC-reactive protein. The product can be administered to a human tocorrect hypothyroidism. The product can be administered to a human tocorrect hyperthyroidism.

In one embodiment, the invention provides for a method of producing aproduct to correct hypercholesterolemia in a human by pulping fruits ofEmblica Officinalis with demineralized water to create a slurry. Theslurry is treated with pectinase to form a pectinase-treated slurry. Thepectinase-treated slurry is filtered to create a solution. The solutionis concentrated to generate the product.

These and other features of the present invention are explained in moredetail in the following non-limiting examples.

Example 1

Five hundred kilograms of fresh amla fruits were pulped with an equalquantity of demineralized water and the slurry was treated with 2 wt %of pectinase enzyme under stirring at room temperature for 6 h and thenfiltered to yield 310 liters of the extract with a solids content of4.8%. This solution was then concentrated below 60° C. to obtain aslurry with a solids content of 15.2%. This was then spray-dried (inlettemperature 180° C., outlet temperature 90° C.) to obtain 13.5 kg of afree flowing powder. The hydrolysable tannin content of this preparationwas at least 30%. The emblicanin A content of this preparation was10.2%.

Example 2 Toxicity Studies

Acute Toxicity

Healthy albino mice of either sex, having body weight 20-25 g were used.They were housed in clean polypropylene cages with food and wateravailable ad libitum. After acclimatization for one week, their bodyweights were recorded and were divided into 8 groups of 6 each. Group Aserved as control and the remaining 7 groups were kept as experimentalgroup. The experimental animals were supplied 200 mg, 400 mg, 600 mg,800 mg, 2.5 g, 5 g and 10 g/kg of amla extract, respectively, orallyafter an overnight fasting. Animals were observed continuously for thefirst 6 h and mortality was recorded for 72 hours.

Amla extract up to a dosage level of 10 g/kg body weight produced noadverse effects such as increased motor activity, tremors, clonicconvulsions, piloerection, muscle spasm, hyperesthesia, ataxia,sedation, hypnosis and analgesia. No mortality was recorded in 72 hours.

Sub Acute Toxicity

Thirty healthy male Sprague-Dawly rats weighing 200-250 g were used forthe present study. They were housed in polypropylene cages (38×23×10cms) with 5 animals per cage and maintained under standard housingconditions (room temperature 24-27° C. and humidity 60-65%) with 12-hlight and dark cycle. The food in the form of dry pellets and water wereavailable ad libitum.

The animal experiments were conducted according to internationallyfollowed ethical standards and approved by the ethics committee of theLittle Flower Hospital and Medical Research Centre, Angamaly, Kerala,India.

The animals were divided into 5 groups of 6 each. Group A served ascontrol while groups B, C, D and E were fed orally a standardizedextract of Emblica extract at dosages of 200 mg, 500 mg, 1.0 g and 2.0 gevery day for 3 months. Body weights were recorded each week. At the endof 3 months blood samples were collected and analyzed for RBC, WBC,haemoglobin (Hb) and lymphocytes. Blood sugar, serum cholesterol, totalprotein, aminotransferases (SGOT, SGPT) and alkaline phosphatase wereestimated by well-established standard methods. Cholesterol contents ofliver and heart were estimated.

At the end of the study, all animals were sacrificed and the variousorgans and tissues were isolated for detailed examination. The mainobservations were:

-   -   1. All animals in the control and experimental groups showed a        steady increase with weight and were in the normal range.    -   2. Haematological and biochemical parameters of both the control        and experimental groups were in the normal range. The inventive        extract was found to enhance the RBC, WBC counts and haemoglobin        in the experimental group to a moderate degree. Three months of        treatment produced a decrease in blood sugar, serum cholesterol        as well as cholesterol in the heart and liver and a moderate        increase in serum total protein.    -   3. Levels of the enzymes SGOT, SGPT and ALP were in the normal        range indicating amla extract had no hepatotoxic effect.    -   4. There was a significant decrease in the HMG CoA reductase        activity in the experimental group in a dose-dependent manner.    -   5. The lumen of aorta, myocardial cells, nephrotic tissues,        hepatocytes, spleen tissue and tissues of the adrenal gland        appeared normal on microscopic examination.    -   6. Oral feeding of amla extract up to a dose of 2 g/kg for 3        months does not produce any toxic effect.

Example 3

Male NZ white rabbits weighing 1.3-1.6 kg were individually caged andfed a normal standard diet. After an acclimatization period, they weredivided into 4 groups of 5 animals each. One group (Group A) served ascontrol and groups B1, B2 and B3 served as experimental groups. Theexperimental groups were made hypercholesterolemic by feeding 100 mgcholesterol along with the diet daily for 4 months. After 4 months,Group B1 was kept as untreated hypercholesterolemic control and theremaining two groups (B2 and B3) were fed orally with amla extract inthe dosage of 10 mg and 20 mg/kg/day, respectively, for additional 4months. Body weights of animals were recorded every 15 days.

Before starting the experiment, fasting blood was collected from allanimals for estimation of serum total cholesterol, LDL cholesterol(LDL-C), HDL cholesterol (HDL-C) and triglycerides (TG). Blood sampleswere also analyzed for haematological parameters (RBC, WBC, haemoglobin(Hb) and lymphocytes). These analyses were repeated every month.

At the end of 8 months, all animals were sacrificed and liver, aorta,spleen, heart, liver and kidney were isolated and examined for grossmacroscopic changes and thereafter fixed in 10% formalin forhistological studies. Tissue cholesterol of liver, kidney, spleen andheart were estimated. A part of the liver was homogenized for estimatingHMG CoA reductase and mevalonate.

One-way ANOVA with repeated measures was used to statistically analyzethe variance over a period of time. Inter-group comparisons were alsomade using the same method. Punnet multiple comparison test was used tocompare the baseline values with periodically observed values. PostANOVA comparison in inter-group analyses was performed by usingTurkey-Kramer multiple comparison test. Paired t-test was used tocompare the biochemical parameters both before and after the experiment.The results are given in the following Tables (1-6).

TABLE 1 Haematological and Biochemical Parameters Group B1 (Hyper- GroupA cholesterolemic Parameters (Control) control) Group B2 Group B3 RBC5.57 ± 0.27 5.63 ± 0.25 5.67 ± 0.21 5.68 ± 0.17 (millions/mm²) WBC 7.63± 0.45 7.2 ± 0.1 7.66 ± 0.21 7.57 ± 0.32 (′000/mm²) Lymphocytes 2.44 ±0.33 2.17 ± 0.08 2.27 ± 0.04 2.61 ± 0.23 (′000/mm²) Hb 14.6 ± 1.21 13.59± 1.0  15.56 ± 1.23  14.4 ± 1.11 (g/dl) Blood sugar 110.6 ± 6.50  118.83± 4.75  102 ± 4.0  104 ± 4.0  (mg/dl) Total protein 5.56 ± 0.77 5.63 ±0.40 5.73 ± 0.30 6.2 ± 0.2 (g/dl) Liver-Chol. 9.43 ± 0.20 14.63 ± 0.51* 9.53 ± 0.10**  9.56 ± 0.18** (mg/g) Heart-Chol. 7.31 ± 0.07  8.51 ±0.20*  7.50 ± 0.10**  7.43 ± 0.10** (mg/g) Kidney-Chol. 5.58 ± 0.10  6.6± 0.18*  5.56 ± 0.08**  5.67 ± 0.01** (mg/g) Spleen-Chol. 3.40 ± 0.103.80 ± 0.12 3.42 ± 0.02 3.32 ± 0.05 (mg/g) HMG CoA to 1.03 ± 0.15 0.90 ±0.2   1.53 ± 0.6**  1.33 ± 0.06** Mevalonate ratio *significant increase(P < 0.05) **Significant decrease (P < 0.05)

TABLE 2 Serum Cholesterol Serum Cholesterol concentration (mg/dl) Group0 Month 4 Month 5 Month 6 Month 7 Month 8 Month A  55.8 ± 5.0  56.66 ±2.7  53.38 ± 3.25  53.33 ± 3.3  54.23 ± 0.9  52.8 ± 1.8 B1   50 ± 2.72229.16 ± 3.2 220.33 ± 2.8 200.83 ± 3.2 185.31 ± 1.9 164.28 ± 3.6 B248.33 ± 4.3 228.13 ± 1.9 163.56 ± 5.8  88.89 ± 5.1  74.57 ± 3.4  63.33 ±2.89 B3 52.49 ± 3.2 230.83 ± 3.2 177.96 ± 4.4  95.0 ± 1.9  72.88 ± 2.0 58.33 ± 2.89

TABLE 3 LDL Cholesterol Serum LDL Cholesterol (mg/dl) Group 0 Month 4Month 5 Month 6 Month 7 Month 8 Month A 39.22 ± 4.91  39.72 ± 2.59  38.6± 5.82  36.51 ± 4.09 37.64 ± 0.53  36.62 ± 1.56 B1 32.38 ± 1.80 195.73 ±4.2 187.36 ± 1.8 169.79 ± 3.6 154.1 ± 3.2 136.22 ± 4.3 B2  32.4 ± 4.68 193.7 ± 2.2  137.5 ± 5.2  67.54 ± 5.61 54.26 ± 2.71  43.03 ± 2.27 B333.57 ± 3.35 195.86 ± 2.4 153.76 ± 5.2  73.82 ± 2.90 53.41 ± 1.43  39.25± 2.12

TABLE 4 VLDL Cholesterol Serum VLDL Cholesterol (mg/dl) Group 0 Month 4Month 5 Month 6 Month 7 Month 8 Month A 8.28 ± 0.76  8.43 ± 0.75  8.08 ±0.44  8.27 ± 0.46  7.95 ± 0.44  7.95 ± 0.44 B1 8.96 ± 0.45 26.94 ± 0.8925.19 ± 1.16  22.3 ± 1.09 20.26 ± 1.14 19.73 ± 0.9 B2 8.79 ± 0.45 26.56± 0.77 17.69 ± 1.41 12.53 ± 0.46 10.25 ± 0.4  9.07 ± 0.46 B3 8.96 ± 0.4526.36 ± 0.79 17.12 ± 0.97  12.2 ± 0.40  9.42 ± 0.39  8.53 ± 0.46

TABLE 5 HDL Cholesterol Serum HDL Cholesterol (mg/dl) Group 0 Month 4Month 5 Month 6 Month 7 Month 8 Month A 8.33 ± 0.43 8.47 ± 0 8.19 ± 08.33 ± 0.45  8.33 ± 0.12  8.24 ± 0.08 B1 8.75 ± 0.83 9.32 ± 0.98 7.78 ±0.82 8.75 ± 0.84  8.94 ± 1.06  8.33 ± 0.13 B2 8.81 ± 1.42 8.05 ± 0.858.19 ± 0 8.89 ± 0.96 10.05 ± 1.76 11.24 ± 1.07 B3 8.81 ± 0.76 8.47 ±1.20 8.61 ± 0.83 8.75 ± 0.84 10.05 ± 1.44 10.62 ± 1.07

TABLE 6 Serum Triglycerides Serum Triglycerides (mg/dl) Group 0 Month 4Month 5 Month 6 Month 7 Month 8 Month A 41.34 ± 3.85  42.15 ± 3.92 40.38 ± 2.22  41.33 ± 2.83  39.74 ± 2.72 39.74 ± 2.72 B1 44.61 ± 2.11134.84 ± 4.4 125.96 ± 5.8 111.53 ± 5.4 121.94 ± 4.3 98.67 ± 4.62 B243.84 ± 0.61 133.33 ± 2.1  88.47 ± 2.15  62.67 ± 2.31  51.28 ± 2.2245.33 ± 2.3 B3 44.61 ± 2.1 132.55 ± 3.3  88.57 ± 4.84  61.0 ± 2.2  47.11± 1.93 42.67 ± 2.3

Results in the above tables reveal the dramatic effect of feeding amlaextract on the lipid profiles of hypercholesterolemic animals. All theparameters returned almost to their original levels after 4 months ofamla treatment. This unprecedented result strongly suggests that withcontinued treatment hypercholesterolemia could be completely reversed,at least in experimental animals.

This reversal of hypercholesterolemia was further supported by theresults of histological examination of the aorta of the animals. Aorticstrips of control groups were normal with normal intima, media andadventia. So was the case with those of amla-treatedhypercholesterolemic rabbits, while there were smooth muscle cellproliferation, fatty infiltration and foam cell formation in theuntreated hypercholesterolemic animals. Hepatocytes of all animalsappeared normal.

Reduced HMG CoA reductase activity was noted in the amla extract-treatedgroups (data not shown). The activity of this key enzyme was reduced by30 and 56%, respectively, in animals of Group B2 and B3.

Example 4 Human Study 1

Hypercholesterolemic subjects (total cholesterol >240 mg/dl, LDLCholesterol >130 mg/dl) of either sex were selected for the study.Patients having valvular heart disease, congestive heart disease anddiabetes and patients taking lipid lowering drugs were excluded from thestudy. A total of 70 patients were enrolled. They were divided intocontrol group (20 patients) and intervention group (50). They werebriefed about the study and written consents were taken beforecommencement of the study. Before commencement of the study bloodsamples were collected from each patient. The intervention group wereadvised to take amla extract in the form of 500 mg hard gelatin capsulesin the dosage of 2-0-2 after meals. The study period was 3 months. Lipidprofiles were determined at the end of each month. Results are given inTables 7 and 8, shown in FIGS. 1 and 2.

Example 5 Human Study 2

Twenty two apparently healthy human volunteers aged 26 to 76 yearsconsumed 500 mg of amla product, referred to as Amlamax, per day. Noneof the participants had a history of myocardial infarction, stroke orcoronary revascularization. Their physical attributes such as height,weight and blood parameters were analyzed before study and 3 monthsafter consumption of AmlaMax. These data are given in Table 9. Theparticipants were arbitrarily divided into three groups, namely thosebetween ages 26 to 45, 46 to 60 and those above 60 years. Response toAmlamax with respect to the studied parameters were impressive (Table10), with only one non-responder each for total cholesterol and LDLcholesterol. There were two non-responders to fasting blood sugarreduction, three did not respond to TG and CRP while 5 persons did notshow the expected increase in their HDL profiles. The extent of responseamong responders (Table 11) were as follows:

-   -   Total cholesterol: (−) 4.6 to 32.3% ↓(mean 13.6%)    -   LDL cholesterol: (−) 4.9 to 41.9% ↓(mean 17.4%)    -   HDL Cholesterol: (+) 2.2 to 44.8% ↑(mean 15.8%)    -   Triglycerides (TG): (−) 1.4 to 62.9% ↓(mean 27.2%)    -   CRP: (−) 4.2 to 65.0% ↓(mean 48.1%)    -   FBS: (−) 1.1 to 28.5% ↓(mean 10.8%)

The magnitude of change generally follows the extent of abnormality inthe respective starting values. For example there were two participantswith very high TG levels of 534 and 350, respectively. These two personsshowed a decrease of 50.4% and 62.9%, respectively, after AmlaMaxtreatment. Similarly the patient showing the highest enhancement of44.8% in HDL cholesterol had the lowest HDL cholesterol to start with.The most significant changes were noted in CRP levels which responded toAmlaMax very well. Here again the person who responded the maximum(88.4% reduction) had the highest starting CRP value of 12.0 which wasreduced by AmlaMax treatment to the normal range of 1.4 mg/L. Theseresults should be considered significant because such grossabnormalities are normally difficult to correct by drug treatment. Theresults on fasting blood sugar (FBS) are also noteworthy. FBS recordedthe least mean changes in the participants, because most of them hadnear-normal FBS values. There was only one diabetic patient (FBS above140 mg/dl (entry No. 1) (Tables 9 and 11)). This patient also recordedthe highest reduction in FBS. AmlaMax did not produce random reductionin blood sugar irrespective of the starting value (as would a normaldrug do) but only in those cases where such a correction is needed andthus does not lead to hypoglycemia, a problem observed with many drugsfor hyperglycemia. One is reminded of one of the three attributes of an‘adaptogen’ (Rasayana in Ayurveda): It must cause only minimal disordersin the body's physiological functions. In other words, AmlaMax correctsonly where correction is required, rightly justifying its position as anadaptogen, and in sharp contrast with common medications.

In agreement with the above observation, AmlaMax also modulated thyroidfunction, correcting both hyper- as well as hypothyroidisms. Among the22 participants involved in the study, there were two persons (one maleand one female) with overt hypothyroidism with TSH levels of 79.96 and111.02 mU/L. These values after 3 months of AmlaMax treatment were 38.96and 76.79 mU/L, respectively and are expected to improve further tonormal values on continued treatment. Similarly, there was one womanpatient with overt hyperthyroidism with TSH level less than 0.01 mU/Lwhich got corrected to normal level (1.32 mU/L) after 3 months ofAmlaMax consumption. These results indicate the normalizing effect ofAmlaMax on thyroid functions in patients with such disorders.

TABLE 9 Effect of the amla product on Lipid profile, CRP and FBS inHealthy Human Volunteers Lipid Profile CRP FBS Chol TG VLDL LDL HDL(mg/l) (mg/dl) Group 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0Mo 3 Mo 0 Mo 3 Mo A 235 200 229 190 46 38 151 125 38 37 2.4 2.3 225 162(Age 275 188 534 265 — 53 — 93 29 42 1.6 2.0 125 94 25-45 Yr) 310 276192 170 38 34 238 204 34 38 4.0 4.5 74 71 (n = 13) 230 211 167 110 33 22156 147 41 42 3.5 2.8 85 76 230 212 158 150 32 30 157 142 41 40 2.8 1.0100 94 270 212 250 250 50 50 173 124 38 47 3.8 2.5 95 94 220 165 196 13039 26 147 100 34 39 1.9 2.1 80 76 240 229 273 180 55 36 147 150 38 432.0 3.2 95 100 235 212 294 185 59 37 142 135 34 40 3.0 1.5 92 76 220 176142 130 28 26 136 99 51 56 4.0 1.9 93 82 235 212 350 130 70 26 121 14739 44 3.0 1.3 110 97 245 224 175 175 35 35 172 147 38 42 2.6 1.1 85 82260 176 158 120 32 24 184 107 44 45 12 1.4 102 76 Group B 210 188 92 7018 14 144 126 48 48 4 1.4 108 147 (Age 275 241 271 210 54 42 180 159 4140 2 1.6 95 88 45-60 Yr) 180 152 192 120 38 24  92 86 42 50 3.8 2.4 7571 (n = 6) 245 224 100 90 20 18 175 164 40 46 3.0 2.9 95 82 285 253 142140 28 28 219 182 38 43 4.5 1.8 100 88 235 259 273 200 55 40 142 181 3838 4.2 1.0 98 97 Group C 220 153 117 80 23 16 163 90 34 47 4.0 1.2 95 88(Age 220 200 242 250 48 50 138 110 34 40 3.7 1.9 90 88 >60 Yr) 245 224167 100 33 20 174 165 38 39 2.9 1.2 125 109 (n = 4) Chol = Totalcholesterol; VLDL = very low density lipoproteins; LDL = low densitylipoproteins; HDL = high density lipoproteins; TG = Triglyceride; CRP =C-reactive protein; FBS = Fasting blood sugar; Mo = Months aftertreatment with AmlaMax

TABLE 10 Response of Healthy Subjects to Treatment with the product Noof Non- Parameter responders Cholesterol 1 (4.5%)  LDL chol 1 (4.5%) HDL chol 5 (22.7%) TG 3 (13.6%) CRP  4 (18.18%) FBS 2 (9.1%)  Totalnumber of subjects treated = 22

TABLE 11 Extent of Response to Treatment with the product in HealthyHuman Subjects C- Fasting LDL HDL reactive blood Subject CholesterolTriglycerides cholesterol cholesterol protein sugar  1 −15% −18.0%−27.2% −2.6% −4.2% −28.0%  2 −31.6 −50.4 — +44.8 +11.1 −24.8  3 −11−11.5 −14.3 +11.7 +12.5 −4.1  4 −8.3 −34.2 −5.8 +2.4 −20.0 −10.6  5 −8.3−5.1 −9.6 −2.5 −64.3 −6.0  6 −21.5 0.0 −28.4 +23.6 −34.3 −1.1  7 −25.0−33.7 −32.0 +14.7 +10.5 −5.0  8 −4.6 −36.1 +2.0 +13.1 +60.0 +5.2  9 −9.8−36.9 −4.9 +17.6 −50.0 −17.4 10 −20.0 −8.5 −27.2 +9.8 −52.5 −11.9 11−9.8 −62.9 +21.5 +12.8 −56.7 −11.9 12 −8.6 0.0 −15.5 +10.5 −57.3 −3.6 13−32.3 −24.1 −41.9 +2.2 −88.4 −25.5 14 −10.5 −24.0 −12.5 0.0 −65.0 +38.615 −12.4 −23.4 −11.7 −2.5 −20.0 −7.4 16 −15.6 −37.5 −6.6 +19.0 −35.9−5.4 17 −8.6 −10.0 −16.9 +15.0 −4.4 −13.7 18 −11.3 −1.4 −6.3 +13.1 −60.0−12.0 19 +10.2 −26.8 +27.4 0.0 −76.2 −1.1 20 −30.5 −31.7 −44.8 +38.2−70.0 −7.4 21 −9.1 +5.4 −20.3 +17.6 −48.7 −2.3 22 −8.6 −40.2 −5.2 +2.6−58.7 −12.8 Mean (−)13.6 (−)27.2 (−)17.4 (+) 15.8 (−)48.1 (−)10.8Change* *Among responders

The foregoing embodiment and advantages are merely exemplary and are notto be construed as limiting the present invention. The present teachingcan be readily applied to other types of apparatuses. Also, thedescription of the embodiments of the present invention is intended tobe illustrative, and not to limit the scope of the claims, and manyalternatives, modifications, and variations will be apparent to thoseskilled in the art.

I claim:
 1. A method of reducing HMG CoA reductase activity comprisingadministering effective doses of an extract of Emblica officinalis to asubject in need thereof, the extract prepared by a method comprising:pulping fruits of Emblica officinalis with demineralized water to createa slurry; treating the slurry with pectinase; filtering the slurry tocreate a solution; and concentrating the solution to generate theextract of Emblica officinalis.
 2. A method for increasing ratio of HMGCoA to mevalonate comprising administering effective doses of an extractof Emblica officinalis to a subject in need thereof, the extractprepared by a method comprising: pulping fruits of Emblica officinaliswith demineralized water to create a slurry; treating the slurry withpectinase; filtering the slurry to create a solution; and concentratingthe solution to generate the extract of Emblica officinalis.